Process and medium for cloning and long-term serial cultivation of human endothelial cells

ABSTRACT

Vigorous growth of endothelial cells from human umbilical vein endothelial cells in vitro is achieved using a culture medium containing endothelial cell growth factor and heparin and/or dextran sulfate. Cell population doubling times of 13 to 21 hours for 60 to 85 populations doublings are obtained, and cloned human endothelial cell strains are established having similar proliferative capacities.

The invention described herein was made in the course of work undergrants or awards from The National Institute of Health, the Departmentof Health and Human Services.

This application is a continuation of Ser. No. 099,241, filed Sept. 21,1987, which is a continuation of both applications Ser. Nos. 848,913 and848,917 both filed Apr. 7, 1986, and both now abandoned which,respectively, are continuations of applications Ser. Nos. 550,305 nowabandoned and 550,306, now abandoned respectively, both filed Nov. 10,1983.

This invention relates to in vitro cultivation of human endothelialcells, and more particularly to a novel culture medium containingheparin and/or a dextran sulfate, and a process employing such mediumfor in vitro cultivation of endothelial cells derived from human bloodvessels by means of which the proliferative lifespan of such cells isgreatly increased.

BACKGROUND OF THE INVENTION

The endothelium forms the luminal surface of the vascular system, and isan integral component in such physiologic functions as wound healing,hemostasis, selective transfer of substances to and from thecirculation, and synthesis of numerous metabolically active compounds.Correspondingly, endothelial involvement is prominent in pathologicconditions including atherosclerosis, diabetes, thrombosis, hemorrhagicdisorders, tumor metastasis, hypersensitivity, and inflammation (Levineet al Biochemical Interactions at the Endothelium, Elsevier, Amsterdam(1983) pp. 313-342). The need for a greater understanding of endothelialfunction has prompted methodological improvements for culturing thiscell in vitro. Bovine endothelial cells have been studied widely due tothe ease with which they can be serially subcultivated (Levine et al,supra; Rosen et al J. Cell Physiol. 107. 123 (1981); Mueller et al,Science 207, 889 (1980)). Human endothelial cells, however, have morefastidious growth requirements and, despite a suggestion that fibroblastgrowth factor and thrombin stimulate their growth, (Gospodarowicz et al,J. Cell Biol. 77, 774 (1978)), little progress has been made in thelong-term serial subcultivation of these cells (Maciag et al , J. CellBiol. 91, 420 (1981); Glassberg et al, In Vitro 18, 859 (1982);(Johnson, J. Clin. Invest 64, 841 (1980); Gimbrone, Jr., et al, J. CellBiol 60, 673 (1974); Jaffe et al, J. Clin. Invest. 52, 2745 (1973);Haudenschild et al, J. Ultrastruct. Res. 50, 22 (1975); Gordon et al, J.Cell Biol. 91, 205A (1981)).

A significant advance in this field was the use of endothelial cellgrowth factor (ECGF) and fibronectin to enhance the replicative capacityof human umbilical vein endothelial (HUVE) cells (Maciag et al., supra).These factors allowed HUVE cells to be subcultivated; however,multiplication was slow (2-3 day doubling time) and these cellsexhibited a short lifespan (27-34 population doublings (PDs)). Humanendothelial cells isolated from adult iliac artery and grown under thesame conditions exhibited an even shorter lifespan (15-18 PDs)(Glassberg et al, supra). In no case have cloned strains of humanendothelial cells been reported.

BRIEF SUMMARY OF THE INVENTION

This invention is predicated on the discovery that heparin and/or adextran sulfate greatly potentiate the stimulatory effect of endothelialcell growth factor on the proliferation of human umbilical veinendothelial cells and of endothelial cells from adult human bloodvessels. It was discovered that by growing cells on a gelatin matrix ina culture medium supplemented by both endothelial cell growth factor andheparin and/or a dextran sulfate, it was possible to subcultivate andclone human endothelial cells from various blood vessels on the order offrom about 60 to 85 population doublings (PDs) with doubling times of 13to 21 hours.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 comprises graphs in which endothelial cell number per cm² isplotted against concentration of endothelial cell growth factor in aculture medium containing 90 μg/ml of heparin (open circles) and anothercontaining no heparin (solid dots). The graphs of the insert in FIG. 1are based on the same data as the larger graphs, but replotted on anexpanded scale for ECGF concentrations between 0 and 25 μg/ml.

FIG. 2 comprises three curves of growth (cell number /cm²) versus timefor three different human umbilical vein endothelial cell linesindicated by open squares, solid squares and open circles, respectively,and

FIG. 3 is a plot of cell density at subculture versus cumulativepopulation doubling level for three separate HUVE cell lines indicatedby solid squares, triangles and dots, respectively, subcultured weekly.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, HUVE cells have been subcultured in culture mediumcontaining endothelial cell growth factor (Maciag et al, supra), butmultiplication was slow (2-3 days doubling time) and short lifespan(27-34 population doublings). Even a shorter life span (15-18 PDs) wasobserved for human endothelial cells from adult iliac artery. It wasdiscovered, however, that if heparin and/or a dextran sulfate areincluded in the culture medium, doubling times were substantiallyreduced to 13 to 21 hours, and 60 to 85 population doublings areobtained before senescence.

The endothelial cell culture medium used in the process of thisinvention comprises any of the well known media for culturing cells,fetal bovine serum, endothelial cell growth factor and heparin and/ordextran sulfate. Of the various cell growth media, Medium 199 (GibcoLaboratories) is particularly preferred. The composition of such mediumis well known (see e.g. Morgan et al, Proc. Soc. Biol. Med, 73, 1(1950)).

In addition to the cell culture medium, such as Medium 199, theendothelial cell culture medium of the invention contains endothelialcell growth factor which is available from Collaborative Research, Inc.ECGF may be prepared as described by Maciag et al, supra. The mode ofpreparation therein described involves preparing hypothalamic extractsby homogenization of bovine hypothalamus followed by stirring of thehomogenate for a period of hours at 4° C., centrifugation and recoveryof the supernatant. The resulting extract is then further fractionatedwith streptomycin sulfate (Gibco) to remove soluble lipid. Thesupernatant is extracted with 0.5% streptomycin sulfate at pH 7.0 for atleast 1 hour, after which the extract is centrifuged at 13,800×g for 40minutes and the supernatant is recovered and can be stored as alyophilized powder at 4° C. The resulting ECGF is an acid-andheat-labile protein, physically and chemically distinct from fibroblastgrowth factor (Gospodarowicz et al, J. Biol. Chem. 253, 3736-3743(1978)), possessing a molecular weight of 75,000 (Maciag et al, Proc.Natl. Acad. Sci. U.S.A., 76, 5674-5678 (1979)).

Heparin has a molecular weight of 6000-25,000 and is aglycosaminoglucuronan (acid mucopolysaccharide) that occurs in mosttissues; human liver, lung and mast cells are especially rich sources.Chemically it is not homogeneous, but is a family of linear polymerswhich differ in chain length and M.W., but have similar disaccharideunits. The disaccharide units are composed of D-glucuronic or L-iduronicacids in 1,4-glycosidic linkage to glucosamine; each unit contains twosulfate esters and one N-sulfate group.

Heparin is obtained commercially from a variety of animal tissues,particularly bovine lung tissue and intestinal mucosa of pigs (seeKirk-Othmer, Encycl. Chem. Tech. 3d 4, pp 14-15 (1978)).

Dextran sulfate is usually available as the sodium salt, and chemicallyis known as dextran sulfuric acid ester sodium salt. It is prepared byboiling dextran with sulfuric acid to reduce the molecular weight andesterifying the degraded dextran with chlorosulfonic acid in pyridine.The molecular weight may vary from about 5000 to about 600,000, and thedextran may contain up to about 25%, by weight, of sulfur. It is a whitepowder, soluble in water and has an activity of about 17 internationalheparin units/mg (see e.g. The Merck Index 9d (1976) page 2911). Thepreferred dextran sulfates for use in the endothelial cell culture mediaof the present invention are one having a molecular weight of about8000, and another having a molecular weight of about 500,000.

The endothelial cell culture medium of this invention will comprise inaddition to a standard cell culture medium, such as Medium 199, fromabout 5 to about 30%, preferably about 20%, by volume of fetal bovineserium, said percentages being based on the total volume of theendothelial cell culture medium. In addition, such novel culture mediumwill contain from about 10 to about 200 μg/ml of endothelial cell growthfactor and from about 9 to about 900 μg/ml of heparin, or about 5 toabout 500 μg/ml of dextran sulfate of MW about 8000, or about 50 toabout 500 μg of a dextran sulfate having a MW of about 500,000. Mixturesof heparin and dextran sulfate may also be used. Preferably, the novelendothelial cell culture medium contains on the order of about 20 μg/mlof ECGF and about 90 μg of heparin, or 100 μg of dextran sulfate.

In carrying out the process of this invention, the endothelial cells areisolated and seeded into gelatin-coated flasks containing theabove-described endothelial cell culture medium. At confluence, thecells are trypsinized and reseeded at desired cell densities. Clones arederived from secondary cultures and seeded at about 10 cells/cm². Theclones are then serially propagated in the said endothelial cell culturemedium.

The below described specific examples, figures and tables further serveto illustrate the invention.

In the case where the endothelial cells are to be obtained from humanvascular tissue, they preferably are obtained from brain-dead butheart-beating cadaver organ, e.g. kidney donors. Under sterileconditions the abdomen of the heart-beating cadaver donor is entered andthe aorta, inferior vena cava (IVC), kidneys and ureters are dissectedout "en bloc." At completion of the dissection, but prior to separationof the organs from the circulatory system and removal, the donor isgiven from at least about 10,000 to 20,000 units, preferably about15,000 units intravenous aqueous heparin and from at least about 5 toabout 15 mg, preferably about 10 mg phenoxybenzamine. The vessels aretransected and the kidneys rapidly cooled with iced saline forpreservation for future transplant. All tissue is then dissected fromthe renal vessels, leaving renal artery and vein and a small patch ofaorta and inferior vena cava. The remaining tissue of the aorta, IVC andiliac vessels, previously discarded in the course of donor nephrectomy,is placed in labelled specimen containers on ice containing theendothelial cell culture medium of this invention, previously described.The amount of each vessel which can routinely be retrieved at this stageis as follows:

    ______________________________________                                        Vessel         Diameter (cm)                                                                             Length (cm)                                        ______________________________________                                        Aorta          1.5-2.5     4-6                                                IVC            2-3         6-8                                                Left iliac artery                                                                            0.8-1.5     4-6                                                Right iliac artery                                                                           0.8-1.5     4-6                                                Left iliac vein                                                                              1-2         4-6                                                Right iliac vein                                                                             1-2         4-6                                                ______________________________________                                    

The time elapsed from heparinization until the specimens are placed incontainers usually does not exceed about 30 minutes, and tissueperfusion usually continues for most or all of that time. The vascularspecimens thus obtained are stored in a cold room. Inasmuch as thesurgery is usually performed at night, the specimens are available foruse the morning following surgery.

Endothelial cells were isolated from a variety of human blood vesselsusing collagenase (Rosen, et al, supra), and seeded into gelatin(1%)-coated flasks containing Medium 199 (Gibco Laboratories) with 20%fetal bovine serum, 20 μg/ml ECGF, and 90 μg/ml heparin At confluence,cultures were trypsinized (0.25% trypsin-0.09% EDTA) and reseeded atdesired cell densities. In addition to the umbilical vein, the donorvessels include the superior mesenteric, iliac, carotid, pulmonary,femoral, and splenic arteries, the thoracic and abdominal aortas, andthe iliac and portal veins. Clones were derived from secondary culturesby seeding at 10 cells/cm² and isolating single cells with glass cloningrings; approximately half the isolated cells grew and were seriallypropagated in the above described medium containing ECGF and heparin.Eleven cloned HUVE cell strains and four abdominal aorta endothelialcell strains were established. All cultures were characterized asendothelial according to morphological and functional criteria(expression of Factor VIII-related antigen (Rosen, et al, supra) andproduction of angiotensin-converting enzyme (Levine et al, supra; Ryanet al Biochem J., 167, 501 (1977)).

Enhanced proliferation and increased lifespan of the human endothelialcell cultures resulted from the inclusion of heparin to the culturemedium. Referring to FIG. 1, there is shown the effect of heparin onHUVE cell growth as a function of ECGF concentration. In the presence ofheparin significant growth was observed with as little as 1 μg/ml ECGFwith maximal growth at 25 μg/ml ECGF; in the absence of heparin, 100-20μg ECGF were required for significant growth. Cultures supplemented withheparin grew to consistently higher densities than those withoutheparin. Heparin at concentrations as high as 900 μg/ml did not supportreplication in the absence of ECGF, demonstrating that both factors wererequired for optimum growth.

FIG. 2 shows growth curves for three lines of HUVE cells cultured with90 μg/ml herparin and 20 μg/ml ECGF; these cultures achieved celldensities at confluence of 10⁵ cells /cm² with doubling times of 17-21hours. In contrast, cells cultured with 100 μg/ml ECGF without heparinwere reported to grow to maximum densities of 4×10⁴ cells/cm² with adoubling time of 64 hours (Maciag et al, supra).

FIG. 3 shows cell densities at confluence throughout the lifespan ofHUVE cells. Proliferative lifespan was determined by measuring harvestcell density at each subculture until proliferation ceased (Rosen et al,supra). The PDs undergone at each subculture were calculated using theformula: PDs=log₂ [(number of cells harvested)/(number of cellsseeded)×(attachment efficiency)], and summed to give the cumulativepopulation doubling level (CPDL). In all three lines harvest celldensities decreased with increasing CPDL as greater numbers of largenondividing cells appeared. Cultures were considered senescent when lessthan one PD occurred within three weeks after subculture.

By this definition, the lifespan of 12 uncloned lines and 11 clonedstrains of HUVE cells ranged from 42 to 85 with a median of 65 CPDLs.Endothelial cells from adult human vascular tissue exhibited shorterdoubling times, and maintained higher harvest cell densities for agreater percentage of their lifespan than HUVE cells. As can be seen byreference to Table 1, lifespan of the cells, in terms of populationdoublings range from 45 to 66.

                                      TABLE 1                                     __________________________________________________________________________                                   Lifespan                                       Culture                                                                              Cell Type                                                                              Age of                                                                            Sex of                                                                            Cause of                                                                             (population                                    Designation                                                                          of Origin                                                                              Donor                                                                             Donor                                                                             Death  doublings)                                     __________________________________________________________________________    Summary of Adult Human Endothelial Cell Lines                                 HAAE-1 Abdominal                                                                              20  M   Head trauma                                                                          64                                                    aorta                                                                  Clones                                                                        HAAE-1B                        45                                             HAAE-1E                        62                                             HAAE-1C                        78                                             HAAE-1F                        67                                             HAAE-2 Abdominal                                                                              51  M   Brain abcess                                                                         61                                                    aorta                                                                  HIVE-2 Iliac vein                                                                             51  M   Brain abcess                                                                         56                                             HFAE-2 Femoral  51  M   Brain abcess                                                                         66                                                    artery                                                                 HPAE-2 Pulmonary                                                                              51  M   Brain abcess                                                                          N.D.*                                                artery                                                                 HPtVE-2                                                                              Portal vein                                                                            51  M   Brain abcess                                                                         N.D.                                           HMAE-2 Superior 51  M   Brain abcess                                                                         N.D.                                                  mesentric artery                                                       HIAE-3 Iliac artery                                                                           32  M   Head trauma                                           Summary of Adult Human Smooth Muscle Cell Lines                               HTAS-2 Thoracic aorta                                                                         51  M   Brain abcess                                                                         N.D.                                           HCAS-2 Carotid artery                                                                         51  M   Brain abcess                                                                         N.D.                                           HAAS-1 Abdominal aorta                                                                        20  M   Head trauma                                           __________________________________________________________________________     *Not determined, frozen at early passage                                 

To confirm that heparin is responsible for stimulating proliferation,the effect of the heparin antagonist, protamine, was examined, and theresults obtained are set forth in Table 2, below.

                  TABLE 2                                                         ______________________________________                                        Effect of Protamine on Human Endothelial Cell Growth                          Additions to Culture Medium.sup.a                                                                  Harvest Cell                                             (μg/ml)           Density.sup.b                                            ECFG    Heparin    Protamine (× 10.sup.4 cells/cm.sup.2)                ______________________________________                                        20      --         --        1.4 + 0.1                                        20        90.sup.c --        5.8 + 0.3                                        20       90         230      1.1 + 0.3                                        20      900         230      6.2 + 0.2                                        200     --         --        5.4 + 0.4                                        200     --         1200       0.7 + 0.01                                      200     990        1200       9.7 + 0.01                                      ______________________________________                                         .sup.a ECGF prepared as described previously (Maciag et al, supra) hepari     (sodium salt, Grade 1, from porcine intestine mucosa; 168 U/mg; Sigma);       protamine sulfate (sodium salt, Grade X, from salmon sperm; Sigma; 150        μg protamine neutralized 100 μg heparin).                               .sup.b Cell counts [+ standard deviation, (n = 3)] seven days after           seeding at 5 × 10.sup.3 cells/cm.sup.2.                                 .sup.c Heparin from bovine lung (sodium salt, U.S.P.; Upjohn).           

As shown in Table 2, stimulation of cell growth by heparin wascompletely blocked by a neutralizing dose of protamine. Growth promotingactivity was reestablished by addition of a tenfold excess of heparin.At high ECGF concentrations (200 μg/ml) significant growth stimulationwas observation in the absence of added heparin. The stimulatoryactivity of 200 μg/ml ECGF was completely blocked by addition of a highdose of protamine (1200 μg/ml); stimulation of cell proliferation wasrestored by addition of excess heparin (990 μg/ml). In addition toheparin, the glycosaminoglycans, chondroiton surface, hyaluronic acidand keratin sulfate, were tested for growth promoting capabilities andhad no effect on endothelial cell growth. The sulphated polysaccharide,dextran sulphate, significantly stimulated proliferation, althoughdextran itself was inactive.

The mechanism by which heparin stimulates human endothelial cellproliferation is unknown. Azizkhan et al, J. Exp. Med. 152, 931 (1980)showed that heparin (and dextran sulfate) increased bovine capillaryendothelial cell migration, but had no effect on proliferation. Otherreports on the effects of heparin on various cell types have yieldedconflicting results (Costachel et al, Exp. Cell Res. 34, 542 (1964);Young et al, Proc. Soc. Exp. Biol. Med 159, 88 (1978); Castellot, Jr. etal, J. Cell Biol. 90, 372 (1981); Lippman, Epithelial-MesenchymalInteractions. 18th Hahnemann Symposium (Williams and Wilkins, Baltimore1968) pp. 208-229). Several investigations have shown that heparin maybind reversibly or irreversibly to the cell surface (Lippman, supra;Kjellen et al, Biochem. Biophys. Res Comm 74, 126 (1977); Glimelius etal, Thromb. Res. 12, 773 (1978)) and, thus, may influence intercellularcommunication (Roblin et al, Biochemistry 14, 347 (1975); Ohnishi, etal, Exp. Cell Res. 93, 136 (1975)) and membrane receptor accessibility(Kraemer et al, Biochem. Biophys. Res. Comm. 56 423 (1974)). Morphologicchanges (Abro et al, Experimentia 31 1453 (1975)) and modifications ofcellular behavior (Azizkhan et al, supra; Regelson, Advances inChemotherapy, Academic Press, New York (1968) Vol. 3, pp 303-370)consistent with cell membrane-heparin interactions have been reported.In vivo, the extracellular matrix of vascular tissue contains highconcentrations of glycosaminoglycans (Gardais et al, Comp. Biochem.Physiol. 44B 507 (1973)). In vitro, heparin-like molecules are secretedby endothelial cells (Buonassisi, Exp. Cell Res. 76, 363 (1973); Busch,et al, Haemostasis 8, 142 (1979); Gamse et al, Biochem Biophys. Acta544, 514 (1978)) and have been shown to inhibit smooth muscle cellgrowth (Castellot et al, supra). The discovery that heparin stimulatesendothelial cell proliferation suggests that heparin-like substances mayplay an important role in cell growth regulation in normal and injuredvessels.

In the past, many basic and applied studies had to be performed onendothelial cells from other animal species because existing culturetechniques permitted only restricted proliferation of human endothelialcells. It is believed that the procedures of this invention describedabove for serial subcultivation can increase the yield of HUVE cells by10⁸ -fold and of adult vessel endothelial cells by 10¹³ -fold overpreviously published methods. This will permit minimal amounts of humanvascular tissue to be used for the generation of large numbers ofcultured endothelial cells, and thus, problems of human pathologyinvolving the endothelium now can be approached directly employing ahuman endothelial cell model. In addition, this cell system should provevaluable for various clinical applications, such as in vitro testing ofvasoactive agents and the coating of artificial graft materials.

I claim:
 1. A culture medium for enhancing proliferation and increasing the lifespan of human endothelial cell cultures which comprises a cell growth medium, and effective amounts of endothelial cell growth factor (ECGF) and a material selected from the group consisting of heparin, dextran sulfate, and mixtures thereof to increase the population doublings of human umbilical vein endothelial cells (HUVE cells) to at least about 42 prior to senescence and to reduce the doubling time of said HUVE cells so as not to exceed about 21 hours.
 2. A culture medium according to claim 1 comprising from about 5% to about 30% fetal bovine serum and from about 70% to about 95% of a cell growth medium, said percentages being by volume based on the total volume of said endothelial cell culture medium.
 3. A cell culture medium according to claim 2 containing from about 10 to about 200 μg/ml of ECGF and from about 9 to about 900 μg/ml of heparin.
 4. A cell culture medium according to claim 3 comprising about 20% fetal bovine serum and containing about 20 μg/ml of ECGF and about 90 μg/ml heparin.
 5. A cell culture medium according to claim 2 containing from about 10 to about 200 μg/ml of ECGF and from about 5 to about 500 μg/ml of dextran sulfate.
 6. A cell culture medium according to claim 5 comprising about 20% fetal calf serum, and containing about 100 μg/ml of dextran sulfate.
 7. A process for enhancing proliferation and increasing the life span of human umbilical endothelial cell cultures which comprises culturing human endothelial cells derived from human umbilical veins in the presence of a cell culture medium containing effective amounts of endothelial cell growth factor (ECGF) and a material selected from the group consisting of heparin, dextran sulfate, and mixtures thereof to increase the population doublings of human umbilical vein endothelial cells (HUVE cells) to at least about 42 prior to senescence and to reduce the doubling time of said HUVE cells so as not to exceed about 21 hours. 